Project Summary Various cellular processes generate reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, which are key mediators of signaling pathways. Dysregulated redox homeostasis and ROS generation are hallmarks of diseases including cancer and age- associated degeneration. Primary cellular targets of ROS are cysteine thiols that form sulfenic, sulfinic, and sulfonic acids, as well as disulfides. ROS generation is localized to specific cellular regions, and are short-lived, necessitating spatiotemporal methods to study cysteine oxidation events. We have developed a chemical-proteomic strategy, termed isoTOP-ABPP, to study diverse oxidative cysteine modifications. To provide the necessary spatiotemporal readout, we will expand our current isoTOP-ABB platform by: (1) utilizing caged cysteine-reactive probes that can be activated rapidly by light for temporal control of cysteine labeling; and, (2) adapting the TurboID proximity biotinylation method to spatially localize our analysis to specific regions of the cell. We will apply these spatiotemporal readouts to two ROS-generating systems in the cell: (1) growth-factor mediated activation of NADPH oxidase (NOX); and, (2) inhibition of the electron transport chain (ETC) and mitochondrial superoxide dismutase. Epidermal growth factor (EGF) binding to EGFR, activates ROS release via NOX2. We aim to identify cysteine oxidation events proximal to NOX2 and characterize the role of these oxidation events on growth-factor signaling. In early studies, we identified a redox-active disulfide bond in a fatty-acid binding protein, FABP5, which is formed upon EGF stimulation of A431 cells. We will investigate the effects of disulfide-bond formation on the lipid-binding properties of FABP5, and downstream signaling pathways mediated by growth-factor stimulation. The mitochondria are highly redox-active organelles, and we aim to provide a comprehensive view of oxidation targets during mitochondrial dysfunction, by selectively enriching mitochondrial proteins prior to analysis. We will apply our optimized mitochondrial methods to interrogate cysteine oxidation in a C. elegans SOD-2 mutant with elevated mitochondrial ROS, and extended lifespan.